It has been reported that many patients, diagnosed as having cancer have elevated serum glycoprotein levels. Such increased glycoprotein levels may be due to increased shedding and/or secretion of glycoproteins by malignant cells or increased host synthesis of glycoproteins in response to a tumor. Efforts to accurately measure such tumor-related increases in total serum glycoprotein levels have been relatively unsuccessful, due in part to the fact that the actual increases in serum glycoprotein levels are small, serum glycoprotein levels vary from individual to individual, and with time, in the same individual.
Carbohydrate acceptors may be derived from tumors, i.e., tumor glycoproteins resulting from necrosis of the cell, or from a host in response to tissue destruction, i.e., produced as a result of tumor growth. Tumor growth provokes the migration of host macrophage and neutrophil cells to the vicinity of the tumor. When the host cells are brought in contact with the tumor cells, degradative enzymes present in host cell lysosomes are released. Lysosomes contain a variety of glycosidases including, for example, sialidase and .beta.-galactosidase, which are capable of producing glycoproteins having terminal N-acetylglucosamine moieties. Other lysosomal glycosidases, such as, N-acetylglucosaminidase, sequentially degrade the entire oligosaccharide group of glycoproteins, thereby creating other terminal carbohydrates. Each of these terminal carbohydrates have the potential to serve as acceptors for the appropriate sugar derivatives and glycosyltransferases. Increased steady state levels of these partially-degraded glycoproteins may be expected in samples obtained from patients having cancer or other chronic diseases due to continuous production of such glycoproteins in the serum of such patients. The increase in the level of partially-degraded glycoproteins may also be due to the inefficient removal of various types of degraded glycoproteins. For example, the turnover or removal of glycoproteins with newly-exposed mannose residues may be slower than the corresponding galactose-terminal glycoproteins, An additional source of glycoproteins with incomplete oligosaccharides may be due to aborted glycosylation of tumor cell glycoproteins. Increased levels of incomplete glycoproteins in serum may result from the destruction of tumor cells by host response and/or by an ineffective system of glycosyltransferases within the tumor cell due to oncogenic transformation. In addition, the host may respond to the presence of a tumor by producing blood glycoproteins which contain sugar acceptor sites.
Although several specific glycoproteins, characteristically found in cancer patients, have been identified, there is no rapid method or procedure for quantitatively determining carbohydrate acceptor subclasses having common oligosaccharide moieties. Davidson, et al, in U.S. Pat. No. 4,146,603 describes a tumor specific glycoprotein characterized as having an isoelectric point of from 4.2 to 4.6 and solubility in perchloric acid. The described tumor specific glycoproteins are detected by mixing a serum sample with perchloric acid to precipitate a fraction and then subjecting the perchloric acid soluble fraction to gel electrophoresis or isoelectric focusing to detect the presence of a tumor specific glycoprotein. Isselbacher, et al, in U.S. Pat. No. 4,261,976 discloses a glycopeptide which inhibits the growth of malignant cells or malignant tumors. The glycopeptide, obtained from animals or humans having malignant cells or tumors, is soluble in phosphotungstic acid and has a molecular weight of about 3600 and is a substrate for isoenzymes of serum galactosyltransferase (GT-I and GT-II). The presence of the glycopeptide is indicated by measuring a known amount of galactosyltransferase in the sample containing the glycopeptide. Plotkin, et al. in U.S. Pat. No. 4,132,600 describes an enzymatic noninvasive method for detecting cancer in mammalian tissue comprising incubation of urine containing exfoliated cells from mammalian tissues suspected of containing cancerous cells and then measuring the galactosyltransferase activity of the cells. Plotkin, et al, in W.O. No. 80/02296 describes a method for determining whether mammalian tissue cells are malignant by assaying cells from said tissues to determine whether the glycoproteins are significantly altered compared to nonmalignant cells. The glycoproteins are indirectly measured by adding to the cells a marker, i.e., a lectin, capable of being detected by nonchemical technique, wherein said marker has a specific affinity for galactose or galactose residue, and thereafter detecting the amount of marker bound to said cells.
It is an object of the present invention to provide a method for directly detecting and quantitatively measuring in a biological sample the level of carbohydrate acceptors, in particular, subclasses of glycoproteins having common oligosaccharide moieties.